Our NCVDG has recently provided the first evidence for the induction of protective immune responses to simian immunodeficiency virus (SIV) in the genital tract of female rhesus macaques. We observed that systemic priming followed by oral or intratracheal boosting with a microencapsulated whole SIV vaccine induced SIV-specific secretory IgA responses in the vagina, and that significant protection was observed against intravaginal challenge with live pathogenic SIV. These results provide the basis for further exploring alternative approaches to develop improved vaccines for mucosal immunization against HIV. In this project a major goal is the production and characterization of non-infectious particulate antigens expressed by recombinant expression vectors, which should be particularly well suited for microencapsulation. Our initial focus here will be on the gag and env genes of the infectious pathogenic molecular clone SIVmac239. We have already constructed a series of baculovirus recombinants and vaccinia virus recombinants expressing the SIV envelope and core proteins, and have investigated conditions for the production and purification of virus-like particles. Alternative cell lines and expression vectors are being employed to obtain particles which contain optimal amounts of envelope proteins. In addition we will explore the use of alternate approaches for mucosal immunization which may have specific advantages including increased duration of the protective immune response. As one additional approach, we will utilize attenuated Salmonella recombinant strains which have been developed for their ability to deliver antigens to B and T lymphocytes in Peyer's patches, thereby priming the mucosal immune system to produce specific immune responses. Antigens to be expressed will include the SIV gp120 protein, an alternating polymer of V3 sequences of the IIIB and MN isolates of HIV-1 (which will enable us to evaluate the potential of a single Salmonella recombinant to induce neutralizing antibody responses to multiple HIV strains) and a construct in which the V3 sequence is fused to the coding sequence for the cholera toxin B subunit (to determine whether CTB functions as a mucosal adjuvant in the context of the Salmonella vector). Finally, the recent discovery that intramuscular injection of plasmid DNA results in the expression of encoded proteins, and that this approach can be used for protective immunization against viral diseases, provides an attractive approach for induction of long term mucosal immune responses. We will utilize our knowledge of mucosal delivery systems to develop effective procedures for immunization of mucosal inductive sites with plasmid vectors encoding SIV and HIV antigens.